The proposed research project focuses a recently uncovered, paradigm-shifting structure-function link relevant to the entire family of trypsin-like enzymes to which thrombin belongs. A pre-existing, allosteric equilibrium between ensembles of closed (E*) and open (E) conformations of the active site influences the level of activity and mechanism of binding in the protease. The equilibrium also exists in the zymogen and explains the spontaneous autoactivation observed with several proteins involved in blood coagulation, immune response, fibrinolysis and development. Observational evidence of the E*-E equilibrium comes from a large body of structures currently deposited in the Protein Data Bank. Additional independent evidence comes from rapid kinetics measurements of ligand binding to the active site of protease and zymogen that support conformational selection as a general mechanism of recognition in the trypsin fold. Studies under specific aim 1 will test the hypothesis that protease and zymogen undergo the E*-E equilibrium in solution and that the relative distribution of E* and E influences activity in the protease and the mechanism of activation in the zymogen. A significant component of these studies will involve pioneering NMR (2D and 19F) measurements of thrombin and prethrombin-2 with the goal of elucidating, for the first time, the structure and dynamics of their free conformation(s) in solution. We will focus on the likely structural determinants of the E*-E equilibrium and critical residues that decorate the entrance to the active site region in the 215-217 segment (W215, G216, E217), the 60-loop (W60d), the autolysis loop (W148) and the 190-193 corridor (E192). The functional role of these residues will be tested by rapid kinetics measurements of ligand binding to the active site of wild-type and mutants of thrombin and its direct zymogen precursor prethrombin-2. These studies will advance our understanding of a basic structure-function link of the trypsin fold and will provide background for studies to be carried out under specific aim 2. Members of the trypsin family of proteases, to which thrombin belongs, are expressed as inactive zymogens and irreversibly converted to the mature protease by proteolytic cleavage at R15 in the activation domain. The cleavage generates a new N-terminus that inserts into the protein core and H-bonds to the side chain of residue D194. Elucidating how the Huber-Bode mechanism of zymogen activation described above is linked to the allosteric E*-E equilibrium will be center stage in our investigation. We will perturb the critical I16- D194 H-bond with several substitutions that weaken or abolish the interaction. Each mutant will be studied by rapid kinetics to directly measure the E*-E distribution in solution. Additionally, key mutants such as D914A will be characterized structurally for the first time by X-ray and NMR to complement studies of prethrombin-2 and thrombin under specific aim 1. Developments from this specific aim will elucidate the linkage between two critical features of the trypsin fold, i.e., the allosteric E*-E equilibrium and the Huber-Bode mechanism, in ways that will advance our basic knowledge of one the largest families of proteases.